Plant Biology ISSN 1435-8603 RESEARCH PAPER Spatial repartition and genetic relationship of green and albino individuals in mixed populations of Cephalanthera orchids V. Tranchida-Lombardo1,2, M. Roy3, E. Bugot3, G. Santoro4,U¨ . Pu¨ ttsepp5, M. Selosse3 & S. Cozzolino2 1 Sezione di Biologia ed Ecologia Vegetale D.A.C.P.A., Universita` di Catania, Italy 2 Dipartimento di Biologia Strutturale e Funzionale, Universita` ‘‘Federico II’’, complesso MSA, Napoli, Italy 3 Centre d’Ecologie Fonctionnelle et Evolutive, CNRS, UMR 5175, Equipe Interactions Biotiques, Montpellier ce´ dex, France 4 Via della Repubblica 37, Bovino (FG), Italy 5 Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, Tartu, Estonia Keywords ABSTRACT Albinos; amplified fragment length polymorphism; mixotrophy; Several green orchids of the Neottieae tribe acquire organic carbon both from their mycoheterotrophy; Neottieae; mycorrhizal fungi and from photosynthesis. This strategy may represent an inter- photosynthesis; spatial repartition. mediate evolutionary step towards mycoheterotrophy of some non-photosynthetic (albino) orchids. Mixed populations of green and albino individuals possibly repre- Correspondence sent a transient evolutionary stage offering opportunities to understand the evolu- S. Cozzolino, Dipartimento di Biologia tion of mycoheterotrophy. In order to understand the emergence of albinos, we strutturale e funzionale, Universita` ‘‘Federico investigated patterns of spatial and genetic relationships among green and albino II’’, complesso MSA, 80126 Napoli, Italy. individuals in three mixed populations of Cephalanthera damasonium and one of E-mail: [email protected] C. longifolia using spatial repartition and Amplified fragment length polymorphism (AFLP) markers. Two of these populations were monitored over two consecutive Editor flowering seasons. In spatial repartition analyses, albino individuals did not aggre- M. van Kleunen gate more than green individuals. Genetic analyses revealed that, in all sampled populations, albino individuals did not represent a unique lineage, and that albinos Received: 23 January 2009; Accepted: 14 July were often closer related to green individuals than to other albinos from the same 2009 population. Genetic and spatial comparison of genets from the 2-year monitoring revealed that: (i) albinos had lower survival than green individuals; (ii) accordingly, doi:10.1111/j.1438-8677.2009.00252.x albinos detected in the first year did not correspond to the those sampled in the second year; and (iii) with one possible exception, all examined albinos did not belong to any green genet from the same and ⁄ or from the previous year, and vice versa. Our results support a scenario of repeated insurgence of the albino pheno- types within the populations, but unsuccessful transition between the two contrast- ing phenotypes. Future studies should try to unravel the genetic and ecological basis of the two phenotypes. convergent evolution that occurred more than 20 times INTRODUCTION (Molvray et al. 2000) and probably derived from a peculiarity Among plants, achlorophyllous species represent fascinating of their seed germination and embryo development. In fact, examples of nutritional adaptation because, by lacking photo- one of the most distinctive orchid characteristics is that their synthetic pigments, they behave as heterotrophs and explore minute seeds contain only minimal reserves of nutrients, and alternative strategies to acquire organic carbon (Graves 1995; depend upon fungi for the resources necessary for germina- Selosse & Roy 2009). To fulfil their carbon requirements, tion and early seedling growth (Rasmussen 1995; Smith & some plant taxa assimilate organic matter directly from a Read 2008). Mycoheterotrophic orchid species retain this photosynthetic source (parasitic plants) or indirectly, with fungus-dependent nutrition at the adult stage and remain help of a fungal partner (Leake 1994). In the latter case, so- non-photosynthetic over their lifetime (Taylor et al. 2002). called ‘mycoheterotrophic’ plants have developed a depen- Mycoheterotrophic orchids have been intensively investi- dence upon adjacent autotrophic plants from which they gain gated in the last decade and, more recently, interest has carbon indirectly by the way of shared mycorrhizal fungal shifted to the intermediate condition in which fungi partly partners (Leake 1994, 2004). This peculiar capacity of obtain- subsidise the nutrition of photosynthetic orchids. These ing carbon heterotrophically is particularly relevant in orch- plants can use fungal C in addition to their photosynthesis, a ids, which comprise approximately 35% of the fully strategy called partial mycoheterotrophy or ‘mixotrophy’ heterotrophic angiosperms (Waterman & Bidartondo 2008). (Selosse & Roy 2009). Although they are demonstrated to Among orchids, the high frequency of mycoheterotrophs is a retain chlorophyll and photosynthesis (Julou et al. 2005; Plant Biology 12 (2010) 659–667 ª 2009 German Botanical Society and The Royal Botanical Society of the Netherlands 659 Tranchida-Lombardo, Roy, Bugot, Santoro, Pu¨ ttsepp, Selosse & Cozzolino Patterns of albino and green Cephalanthera spp Girlanda et al. 2006), mixotrophic orchids have an isotopic and albino phenotype. The phenotype does not change in composition (13C and 15N) more or less close to that of my- Cephalanthera spp. populations for up to 12 years (Julou et coheterotrophic plants due to the use of fungal organic mat- al. 2005; Abadie et al. 2006), which could reflect genetic, but ter (Gebauer & Meyer 2003; Julou et al. 2005; Abadie et al. also micro-environmental determinism of the phenotype. 2006). This occurs in a number of forest-dwelling species, Thus, spatial and genetic investigations may contribute to especially orchids that were traditionally considered as obli- understanding the origin(s) of albino genotypes in mixed gate autotrophs (Gebauer & Meyer 2003; Bidartondo et al. populations and their genetic correlation with other green 2004; Selosse et al. 2004; Julou et al. 2005). From an evolu- and albino individuals. In particular, it would be interesting tionary point of view, mixotrophy is viewed as a predisposi- to gather information on the following questions: tion to the evolution of mycoheterotrophy (Abadie et al. Do albino individuals represent a unique lineage within 2006; Selosse & Roy 2009), as it occurs in several orchid taxa each mixed population? Are the albino individuals more clo- where mycoheterotrophy secondarily evolved. Among those, sely related to each other than to the green individuals? the tribe Neottieae revealed to be especially pre-adapted to Do some albino individuals belong to the same genet as mycoheterotrophy by virtue of the association of green spe- green individuals? Are there temporal transitions of the same cies with ectomycorrhizal fungi that link them to nearby trees genet between green and albino phenotype or vice versa? (Bidartondo et al. 2004; Selosse et al. 2004; Julou et al. 2005). How are the albino individuals spatially distributed in pop- Investigation of those intermediate mixotrophic stages, ulations? Are albino individuals spatially closer to each other particularly at population level, may provide a unique oppor- than to the green individuals? tunity to understand the evolutionary transitions to mycohet- To answer these specific questions, we investigated the pat- erotrophy. In this regard, achlorophyllous (= ‘albino’) terns of spatial and genetic relationships among green and individuals that are observed in otherwise green species, such albino individuals in three mixed populations of Cephalan- as Epipactis and Cephalanthera spp. (Salmia 1986, 1989), are thera damasonium and one of C. longifolia using spatial of particular interest (Fig. 1). Being themselves mycohetero- repartition and markers. trophic (Julou et al. 2005; Abadie et al. 2006), these albinos may document an evolutionary transition from mixotrophy to mycoheterotrophy. Studies on mixed populations of albino MATERIALS AND METHODS and green individuals have been particularly focused on the Plant models and sampling comparison of C and N sources, fungal specificity, photosyn- thetic capability and morphology, comparing albino and Cephalanthera damasonium (Miller) Druce and C. longifolia green individuals in order to detect pathways of this transi- (L.) Fritsch are two closely related species belonging to the tion. In Epipactis microphylla as well as in Cephalanthera tribe Neottieae (Pridgeon et al. 2008). These species are widely damasonium and C. longifolia, albino individuals do not distributed in the Eurasian temperate zone, and mainly grow differ in their community of fungal symbionts from green on calcareous soil (Delforge 1995). We focused on three natu- individuals (Selosse et al. 2004; Julou et al. 2005; Abadie et al. ral populations of C. damasonium and one of C. longifolia 2006), suggesting a potentially easy shift between the green (see Table 1 for locations and abbreviations). The population of C. longifolia had previously been investigated by Abadie et al. (2006) for mycorrhizal associations and C metabolism. A subsample of each population was examined in genetic analy- ses. Healthy leaves were collected from a total of 155 individu- als (115 green and 40 albinos) and quickly stored in silica gel or frozen at )80 °C. For the Bov C. damasonium population, we replicated the sampling for genetic analysis in two follow- ing years (May 2005 and 2006); here, all